One-step Generation of Phenotype-expressing Triple-knockout Mice with Heritable Mutated Alleles by the CRISPR/Cas9 System

Triple-knockout mice generated by the one-step CRISPR/Cas9 system were examined for the effects of multiple gene modifications on each phenotype and individual gene function. Sixty embryos were transferred, and 9 pups were obtained; all 9 pups had mutations on 3 loci, and 7 pups showed mutations in all-alleles. F0 mice showed knockout phenotypes or no protein expression of target genes simultaneously, and these mutations were normally inherited in the next generation.     

Structural characterization of the trisaccharide chain from the skin mucus glycoprotein of the rainbow trout, Salmo gairdneri

Skin mucus glycoprotein (RGP) was extracted from the skin mucus of rainbow trout, Salmo gairdneri. Alkaline borohydride treatment of RGP yielded a major oligosaccharide alditol NeuAcα2 to6GalNAc-ol and several minor oligosaccharide alditols. A trisaccharide alditol (OS-I) was purified by Sephadex G-25 gel filtration and high-performance liquid chromatography (HPLC). The amount of OS-I was estimated to be 7% in RGP from the amount of GalNAc-ol. Structural determination of the OS-I was examined. Structure of OS-I was identified to be NeuAcα2 to6 (GalNAcα1 to3) GalNAc-ol by α-N-acetylgalactosaminidase digestion, methylation analysis, and ¹H-NMR spectroscopy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Prevalence of zoonotic Bartonella species among rodents and shrews in Thailand

We investigated the prevalence of Bartonella species in 10 rodent and one shrew species in Thailand. From February 2008 to May 2010, a total of 375 small animals were captured in 9 provinces in Thailand. Bartonella strains were isolated from 57 rodents (54 from Rattus species and 3 from Bandicota indica) and one shrew (Suncus murinus) in 7 of the 9 provinces, and identified to the species level. Sequence analysis of the citrate synthase and RNA polymerase β subunit genes identified the 58 isolates from each Bartonella-positive animal as B. tribocorum in 27 (46.6%) animals, B. rattimassiliensis in 17 (29.3%) animals, B. elizabethae in 10 (17.2%) animals and B. queenslandensis in 4 (6.9%) animals. R. norvegicus, R. rattus, and Suncus murinus carried B. elizabethae, which causes endocarditis in humans. The prevalence of Bartonella bacteremic animals by province was 42.9% of the animals collected in Phang Nga, 26.8% in Chiang Rai, 20.4% in Sa Kaeo, 16.7% in Nakhon Si Thammarat, 12.0% in Surat Thani, 9.1% in Mae Hong Son and Loei Provinces.     

Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

Effects of elevated CO2 concentration on growth, annual ring structure and photosynthesis in Larix kaempferi seedlings

We evaluated the effects of elevated carbon dioxide concentration ([CO2]) and two nutrient regimes on stem growth rate, annual ring structure and temporal variations in photosynthetic characteristics of seedlings of Japanese larch (Larix kaempferi (Lamb.) Carr.). Seedlings were grown in phytotron chambers in an ambient (360 ppm) or an elevated (720 ppm) [CO2] in two nutrient regimes for one growing season. Elevated [CO2] reduced stem height and increased stem basal diameter compared with ambient [CO2]. The effect of elevated [CO2] on growth tended to be greater at high-nutrient supply than at low-nutrient supply. Elevated [CO2] had no significant effect on ring width or the number of tracheids per radial file. There was no obvious difference in cell wall thickness or the relative area of the cell wall between seedlings grown in ambient or elevated [CO2]. Although growth in elevated [CO2] resulted in a slight increase in cell diameter, the increase had a relatively minor effect on the relative area of the cell wall. Net assimilation rate increased in response to elevated [CO2]; however, the increase in whole-crown photosynthetic rate (Total Agrowth) in seedlings in the elevated [CO2] treatment was minimal because of the smaller specific needle area and acclimation of the photosynthetic characteristics of the needles to the growth [CO2]. In conclusion, we observed no obvious enhancement in the capacity for carbon fixation in Japanese larch seedlings grown in the presence of elevated [CO2] that might be attributable to changes in stem growth. However, elevated [CO2] caused changes in the temporal pattern of stem growth and in some anatomical features of the tracheids.     

Reduction of aluminum toxicity to radish by alkaline oxygen treated kraft lignin

To obtain a soil-conditioning agent for acid soil containing excess aluminum ions (AL), kraft lignin was modified by alkaline oxygen treatment. The growth of radish root in solution and in soil containing AL with or without addition of these lignins under controlled pH was examined. We concluded the following. Growth inhibition of radish roots by AL can be removed by adding alkaline oxygentreated lignins in the range of pH 4.5–4.8 in soil. A similar result was obtained at pH 4.5 in a culture solution. The reduction of AL toxicity to plant may be due to the aggregation between AL and the modified lignin at low concentrations of modified lignin because soluble AL could not be detected. On other hand, elongation of radish root was not obviously inhibited, although the soluble AL in the solution culture was at an extremely high level when the dosage of a modified lignin was high. This suggests that the reduction in AL toxicity to plants was due to formation of a complex between AL and acidic groups of the modified lignin.Part of this report was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999 and the 44th Lignin Symposium, Gifu, October 1999     

Growth and annual ring structure of Larix sibirica grown at different carbon dioxide concentrations and nutrient supply rates

We compared effects of ambient (360 vpm) and elevated (720 vpm) carbon dioxide concentration ([CO2]) and high and low nutrient supply rates on stem growth, annual ring structure and tracheid anatomy of Siberian larch (Larix sibirica Ledeb.) seedlings over two growing seasons. Elevated [CO2] had no significant effect on either stem height or diameter growth; however, both stem height and diameter growth were enhanced by the high nutrient supply rate, and these increases were stimulated by elevated [CO2]. Elevated [CO2] tended to increase the width of the annual xylem ring, the number of cells in a radial file spanning the ring, and tracheid lumen diameter, whereas it tended to reduce cell wall thickness, although there were no statistically significant CO2 effects on tracheid anatomy. Changes in tracheid cell morphology seemed to be dependent on changes in shoot elongation rates.     

Analysis of blood clotting with the total thrombus analysis system in healthy dogs

To date, coagulation tests are unable to reflect in vivo coagulation status in the same system, including platelet function, fibrin clot formation, and whole blood flow. The Total Thrombus Analysis System (T-TAS), which is a microfluidic assay that simulates conditions in vivo, measures whole blood flow at defined shear rates under conditions designed to assess platelet function (PL-chip) or coagulation and fibrin clot formation (AR-chip). The T-TAS records occlusion start time, occlusion time, and area under the curve. We evaluated this test in healthy control dogs. We also investigated the effect in vivo of acetylsalicylic acid (ASA), and the effect in vitro of an anticoagulation drug (dalteparin; low-molecular-weight heparin; LMWH). The CV of the AUC of both chips was good (CVs of 6.45% [PL] and 1.57% [AR]). The inhibition of platelet function by ASA was evident in the right-shift in the PL test pressure curve. The right-shift in the AR test pressure curves showed that the administration of LMWH inhibited both platelets and the coagulation cascade. The T-TAS may be useful in the evaluation of canine blood coagulation.     

Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins

Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.     

Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.